Persistent infection with high-oncogenic risk human papillomavirus (HR-HPV) types is recognized as a necessary, but not sufficient, cause of invasive carcinoma of the cervix (ICC) [1-3]. HPVs 16 and 18 are the most prevalent types in malignant lesions, accounting for over 70% of ICC and over 50% of high-grade precursor lesions. The HR-HPV E6 and E7 proteins are consistently expressed in dysplasias and carcinomas, disrupting the cell cycle regulatory proteins p53 and pRb, respectively. The obligatory expression of E6 and E7 by both dysplastic and invasive malignant lesions, as well as the viral origin of these proteins, make them excellent targets for HPV therapeutic vaccines.
Listeria monocytogenes (Lm) is a food-borne gram-positive bacterium that can occasionally cause disease in humans, in particular elderly individuals, newborns, pregnant women and immunocompromised individuals. In addition to strongly activating innate immunity and inducing a cytokine response that enhances antigen-presenting cell (APC) function, Lm has the ability to replicate in the cytosol of APCs after escaping from the phagolysosome, mainly through the action of the listeriolysin O (LLO) protein. This unique intracellular life cycle allows antigens secreted by Lm to be processed and presented in the context of both MHC class I and II molecules, resulting in potent cytotoxic CD8+ and Th1 CD4+ T-cell-mediated immune responses. Lm has been extensively investigated as a vector for cancer immunotherapy in pre-clinical models Immunization of mice with Lm-LLO-E7 induces regression of established tumors expressing E7 and confers long-term protection. The therapeutic efficacy of Lm-LLO-E7 correlates with its ability to induce E7-specific CTLs that infiltrate the tumor site, mature dendritic cells, reduce the number of intratumoral regulatory CD4+ CD25+ T cells and inhibit tumor angiogenesis.
Lm has also a number of inherent advantages as a vaccine vector. The bacterium grows very efficiently in vitro without special requirements and it lacks LPS, which is a major toxicity factor in gram-negative bacteria, such as Salmonella. Genetically attenuated Lm vectors also offer additional safety as they can be readily eliminated with antibiotics, in case of serious adverse effects and unlike some viral vectors, no integration of genetic material into the host genome occurs. However, there is always great concern about the safety of a live bacterial vaccine such as Lm, especially regarding its mechanism of attenuation.
The PrfA protein controls the expression of a regulon comprising essential virulence genes required by Lm to colonize its vertebrate hosts; hence the prfA mutation strongly impairs PrfA ability to activate expression of PrfA-dependent virulence genes. The present invention addresses this concern by providing a prfA mutant Listeria that carries a mutant prfA (D133V) gene in the pGG55 plasmid that restores partial PrfA function.